Synapses are the junctions through which neurons communicate with each other and transmit signals that control our development, movements and cognitive abilities. An understanding of the molecular steps involved in the development of synapses in the brain is critical for an understanding of disorders of human cognition that may arise when the processes of synapse formation, maintenance and refinement go awry. However, due to the complexity of the temporal and spatial orchestration of the many components of synapses, it has been difficult to distinguish specific steps in the process and to analyze the individual contributions of specific molecules. Our laboratory has shown that members of the EphB family of receptor tyrosine kinases and their ephrinB ligands are involved in the clustering of specific molecules at the excitatory synapse that result in calcium influx leading to depolarization. However, the molecular mechanisms by which these receptor/ligand complexes promote their effect remain unclear. The objectives of this proposal are use a chemical genetic approach to gain a better understanding of the mechanisms by which EphB/ephrinB signaling controls synapse development. To this end, we have generated a mouse strain with knock-in mutations in the kinase domains of all three EphBs that allows for precise temporal control of the kinase activities of the receptors. Using these mice, we can distinguish between the kinase-dependent and kinase-independent events that occur at the synapse. We have also identified a guanine nucleotide exchange factor that is activated by EphB/ephrinB signaling. We propose to analyze the role of this downstream effector in the formation of synapses and in spine development. For these studies, we propose the following two Specific Aims: 1) To characterize the mechanisms by which the EphB receptor tyrosine kinase regulates excitatory synapse formation, maturation and plasticity; 2) To study the role of a novel guanine nucleotide exchange factor Ephexin5 in EphB-dependent synapse development and maturation. These studies have the potential to not only provide insights into the molecular events that shape synapse development, but also how the dysfunction of these events may lead to diseases of human cognition.During mammalian development, synaptic activity shapes the formation and maintenance of the trillions of neuronal connections that constitute the circuitry of the brain. Many disorders of human cognition including autism, epilepsy and neurodegenerative diseases are caused by defects in the molecular events that regulate the formation, maintenance and refinement of synapses. An understanding of the molecular steps that control synapse development will provide insights into how their dysregulation gives rise to a disease of human cognitive function, and may ultimately suggest therapies for treatment and/or prevention of these disorders.